The use of transgenic mosquitoes in an integrated malaria control strategy will require mosquitoes that block parasite development and remain competitive with wild mosquito populations. Manipulation of key signaling cascades that regulate both immunity and fitness, such as the insulin/insulin growth factor signaling (IIS) cascade, represent a novel approach to achieving this goal. In mosquitoes and other model invertebrates the midgut functions as a center for IIS control of immunity, lifespan, metabolism, and reproduction. The effects of midgut IIS are largely mediated through mitochondrial dynamics, defined as the sum of mitochondrial biogenesis and clearance of damaged mitochondria through mitophagy. In invertebrates and mammals, IIS- dependent mitochondrial dynamics and, hence, mitochondrial metabolism regulate a wide range of important physiologies, including epithelial barrier integrity, stem cell maintenance and differentiation, lifespan and immunity, indicating that this regulation is fundamental in living organisms. Through our work with Anopheles stephensi, we have discovered that manipulation of IIS in the midgut alters the critical balance of mitochondrial biogenesis and mitophagy, resulting in phenotypic changes to mosquito resistance to Plasmodium falciparum infection, as well as mosquito lifespan and reproduction. Thus, we propose that IIS-dependent mitochondrial dynamics control A. stephensi midgut health, which underlies the effects of IIS on immunity, lifespan, metabolism, and reproduction. To define how IIS-dependent mitochondrial dynamics regulate these important phenotypes, we will use five distinct treatments (Akt transgenic (TG), PTEN TG, insulin-fed, IGF1-fed, and manipulation of A. stephensi insulin-like peptides (AsILPs)) to push and pull mitochondrial dynamics in the midgut. This will allow us to identify and manipulate specific gene targets downstream of the IIS cascade that retain malaria parasite resistance while concurrently enhancing midgut health and overall mosquito fitness. To accomplish this, we will first define how midgut IIS regulates mitochondrial biogenesis and clearance through mitophagy and the impacts of these processes on energy homeostasis, stem cell maintenance and differentiation, epithelial integrity, and cell death processes. Since our four exogenous treatments (Akt TG, PTEN TG, insulin-fed, IGF1-fed) impact AsILP transcript expression in the midgut in predictable patterns, AsILPs likely function as natural mediators of IIS-dependent midgut mitochondrial dynamics. As such, we will manipulate AsILPs to alter midgut mitochondrial dynamics and to further clarify this association with IIS- dependent control of P. falciparum infection. Based on associations of IIS-dependent mitochondrial dynamics with mosquito fitness and resistance to parasite infection, we will identify candidate genes that optimally control midgut mitochondrial dynamics to specifically enhance these phenotypes. To this end, we will overexpress or disrupt expression of candidate genes in the midgut using a variety of tools, with the ultimate goal of generating stably transformed, fit A. stephensi that ar completely resistant to P. falciparum infection.